Budding yeast (Saccharomyces cerevisiae) origin recognition complex (ORC) requires ATP to bind specific DNA sequences, whereas fission yeast (Schizosaccharomyces pombe) ORC binds to specific, asymmetric A:T-rich sites within replication origins, independently of ATP, and frog (Xenopus laevis) ORC seems to bind DNA non-specifically. Here we show that despite these differences, ORCs are functionally conserved. Firstly, SpOrc1, SpOrc4 and SpOrc5, like those from other eukaryotes, bound ATP and exhibited ATPase activity, suggesting that ATP is required for pre-replication complex (pre-RC) assembly rather than origin specificity. Secondly, SpOrc4, which is solely responsible for binding SpORC to DNA, inhibited up to 70% of XlORC-dependent DNA replication in Xenopus egg extract by preventing XlORC from binding to chromatin and assembling pre-RCs. Chromatin-bound SpOrc4 was located at AT-rich sequences. XlORC in egg extract bound preferentially to asymmetric A:T-sequences in either bare DNA or in sperm chromatin, and it recruited XlCdc6 and XlMcm proteins to these sequences. These results reveal that XlORC initiates DNA replication preferentially at the same or similar sites to those targeted in fission yeast. The eukaryotic origin recognition complex (ORC) selects the genomic sites where pre-replication complexes are assembled and DNA replication begins. In proliferating mammalian cells, ORC activity appears to be regulated by reducing the affinity of the Orc1 subunit for chromatin during S-phase, and then preventing reformation of a stable ORC/chromatin complex until mitosis is completed, and a nuclear membrane is assembled. Here we show that part of the mechanism by which this is accomplished is through the selective association of Orc1 with Cdk1(Cdc2)/cyclin A during the G2/Mphase of cell division. This association accounted for the appearance of hyperphosphorylated Orc1 in M-phase cells that was subsequently dephosphorylated during the M to G1 transition. Moreover, inhibition of Cdk activity in metaphase cells resulted in rapid binding of Orc1 to chromatin. However, chromatin binding was not mediated through increased affinity of Orc1 for Orc2, suggesting that additional events are involved in the assembly of functional ORC/chromatin sites. These results reveal that the same cyclin-dependent protein kinase that initiates mitosis in mammalian cells also concomitantly inhibits assembly of functional ORC/chromatin sites.